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Y Takata

Publications and source records attributed to Y Takata.

At least 91 records · Page 5Linked to original sources
PubMed1998

Expression of cathepsin B-like enzyme activity in cell lysate from cultured human normal keratinocytes.

In the present study, we have found that the cell lysate from cultured human normal keratinocytes from foreskin (HFKs) hydrolyzed alpha-N-benzoyl-DL-arginine beta-naphthylamide (BANA), and the BANA hydrolysis occurred most under conditions of 37 degrees C and pH 6.0. This activity was strongly inhibited by leupeptin, which is an inhibitor to cathepsin B. These results suggested that the cell lysate from cultured HFKs contained cathepsin B-like enzyme activity. This is the first report to demonstrate that cathepsin B-like enzyme activity was expressed in the cell lysate from human normal keratinocytes.

Benzoylarginine-2-Naphthylamide↗
PubMed1997

Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine.

An expression vector was constructed that produced rat glycine N-methyltransferase in Escherichia coli. Recombinant glycine N-methyltransferase was purified to homogeneity by DEAE-cellulose and gel-filtration chromatography, with a yield of more than 80 mg of pure enzyme from a 1 litre culture. HPLC of tryptic peptides and analysis of isolated peptides showed that the recombinant enzyme was structurally identical with the liver enzyme except for the absence of N-terminal blocking. The alpha-amino group of rat glycine N-methyltransferase is blocked by acetylation [Ogawa, Konishi, Takata, Nakashima and Fujioka (1987) Eur. J. Biochem. 168, 141-151]. In contrast with the liver enzyme, which shows sigmoidal kinetics toward S-adenosylmethionine at all pH values tested [Ogawa and Fujioka (1982) J. Biol. Chem. 257, 3447-3452], the recombinant enzyme exhibited hyperbolic kinetics at low pH and sigmoidal rate behaviour at high pH. The Hill coefficient increased with increasing pH and a pKa of 8.11 was obtained in this transition. The values of Vmax and Km for glycine were not different between the two enzymes. These results suggest that elimination of the positive charge at the N-terminal end either by acetylation or deprotonation is required for co-operative behaviour.

Amino Acid Sequence↗
PubMed1997

Disrupted cholecystokinin type-A receptor (CCKAR) gene in OLETF rats.

OLETF rats develop hyperglycemia, hyperinsulinemia and mild obesity, which is characteristic of human non-insulin-dependent diabetes mellitus (NIDDM). We cloned and sequenced the cholecystokinin type-A receptor (CCKAR) gene in the rats. Comparing the DNA sequences of the OLETF CCKAR gene and LETO CCKAR gene, normal gene, we found a deletion in the OLETF gene, 6847 bases in length, which was flanked by two 3-base-pair direct repeats (5'-TGT-3') at positions -2407/-2405 and 4441/4443, numbered according to the LETO gene sequence, one of which was lost. The promoter region, the first and second exons were missing in the mutant. The region upstream and downstream of the deletion, including exons 3, 4 and 5, was conserved between the two strains, and did not contain any base changes. We found that the gene mapped to chromosome 14 in rats. OLETF rats are the naturally occurring knockout animals with the homozygously disrupted CCKAR gene.

Animals↗
PubMed1997

Functional importance of heat shock protein 90 associated with insulin receptor on insulin-stimulated mitogenesis.

The role of stress proteins on the function of insulin receptor is not well understood. In the rat-1 fibroblasts overexpressing human insulin receptors, heat shock protein (Hsp) 90 was co-immunoprecipitated with insulin receptors and the association was not affected by insulin stimulation. A GST-fusion protein containing the intracellular insulin receptor beta subunit was associated with Hsp 90 in vitro, suggesting the direct interaction of this protein with insulin receptor beta-subunit. Furthermore, microinjection of anti-Hsp 90 antibody into these cells completely inhibited insulin-stimulated mitogenesis. However, neither epidermal growth factor-stimulated nor serum-stimulated mitogenic signal in the cells was affected by the antibody microinjection. These results suggest that Hsp 90 constitutively binds to insulin receptor beta-subunit, which may be necessary for insulin signaling in mitogenesis.

Animals↗
PubMed1997

Functional importance of Shc tyrosine 317 on insulin signaling in Rat1 fibroblasts expressing insulin receptors.

Shc is phosphorylated on Tyr-317, which serves as a docking site for Grb2. To investigate the specific role of Shc phosphorylation and Shc.Grb2 coupling on insulin signaling, we generated expression vectors for wild-type (WT-Shc) and a mutant Shc with a Tyr-317 --> Phe substitution (317Y/F-Shc) and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). From different clonal cell lines, cells expressing 10 times greater amounts of WT-Shc or 317Y/F-Shc compared with endogenous Shc were chosen for analysis of insulin signaling. Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in WT-Shc cells. Because of competition between Shc and IRS-1 for interaction with the insulin receptor, insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in WT-Shc cells compared with that in HIRc cells. Likewise, reduction of endogenous Shc expression by antisense Shc mRNA resulted in increased insulin stimulation of IRS-1 phosphorylation. Although 317Y/F-Shc was also able to interact with insulin receptor, decreased amounts of Shc phosphorylation and Shc association with Grb2 were observed in 317Y/F-Shc cells, indicating that 317Y/F-Shc functions as a dominant-negative mutant. The kinetics of mitogen-activated protein (MAP) kinase activation closely paralleled the kinetics of Shc phosphorylation. Thus, insulin stimulation of MAP kinase activation occurred more rapidly and was prolonged in WT-Shc cells, while the activation was delayed and transient in 317Y/F-Shc cells compared with that in HIRc cells. Importantly, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine and bromodeoxyuridine incorporation, whereas the sensitivity was decreased in 317Y/F-Shc cells. These results indicate that Shc Tyr-317 phosphorylation plays an important role, via coupling with Grb2 and competition with IRS-1, in signal transduction to MAP kinase by insulin, ultimately leading to mitogenesis in Rat1 fibroblasts.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Mouse cholecystokinin type-A receptor gene and its structural analysis.

The mouse cholecystokinin type-A receptor (CCK(A)R) gene was cloned and sequenced, and the exon/intron boundaries were determined by cDNA cloning. The gene, approximately 10 kb in length, contains the entire coding region, and consists of five exons. The deduced amino acid sequence was homologous with that of other species, with the exception of an additional DNA sequence encoding 7 amino acids in exon 5. A region of the 5' end of exon 2 appeared to be alternatively spliced, and generated an isoform shorter by 52 bases. The shorter isoform may encode an 48 amino acid open reading frame due to frameshift of translation. These two mRNA isoforms were expressed equally in the mouse gallbladders.

Alternative Splicing↗
PubMed1997

Fatty acid induced insulin resistance in rat-1 fibroblasts overexpressing human insulin receptors: impaired insulin-stimulated mitogen-activated protein kinase activity.

Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated nonesterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1-4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu4:Tyr1), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7% that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Ambulatory blood pressure monitoring in patients with essential hypertension treated with a new calcium antagonist, cilnidipine.

Cilnidipine (FRC-8653), a new dihydropyridine calcium antagonist, was given to 14 hospitalized patients with essential hypertension, and 24-hour ambulatory blood pressure (BP) monitoring was performed. Once-daily administration of cilnidipine (5-20 mg) for 1-3 weeks decreased the 24-hour average BP significantly from 149 +/- 4/88 +/- 2 mmHg to 141 +/- 3/82 +/- 2 mmHg without any change in the pulse rate. The decrease in ambulatory BP by cilnidipine was evident during the daytime (156 +/- 4/93 +/- 2 mmHg to 143 +/- 5/84 +/- 2 mmHg, p < 0.01 for systolic BP and p < 0.01 for diastolic BP), while it was mild during nighttime (141 +/- 4/80 +/- 2 mmHg to 133 +/- 4/76 +/- 3 mmHg, p < 0.05 for systolic and ns for diastolic BP). The decrease in the ambulatory BP over the whole day and during the nighttime was significantly correlated with the basal ambulatory BP levels. When the subjects were divided into the high ambulatory BP (n = 7) and low ambulatory BP (n = 7) groups, the BP reduction by cilnidipine was evident throughout 24 hours in the high ambulatory BP group, while it was mild and significant only during daytime in the low ambulatory BP group. In summary, once-daily cilnidipine exerts a sufficient and prolonged reduction of BP without an increase in the pulse rate in patients with hypertension. The potency of cilnidipine to decrease ambulatory BP may depend on the basal ambulatory BP level. Cilnidipine is thus a useful antihypertensive drug that may not cause an excessive decrease in BP or a reflex tachycardia.

Adult↗
PubMed1997

Role of eicosanoids in alteration of membrane electrical properties in isolated mesenteric arteries of salt-loaded, Dahl salt-sensitive rats.

1. The role of eicosanoids in altered membrane electrical properties of Dahl salt-sensitive (DS) rats was investigated, by use of conventional microelectrodes technique, in isolated superior mesenteric arteries of DS rats and Dahl salt-resistant (DR) rats fed either a high or low salt diet. 2. The membrane was significantly depolarized in salt-loaded DS rats compared with the other three groups. In addition, the arteries of salt-loaded DS rats exhibited spontaneous electrical activity. 3. Spontaneous electrical activity in salt-loaded DS rats was inhibited by the following: indomethacin, a cyclo-oxygenase inhibitor; ONO-3708, a prostaglandin H2/thromboxane A2 receptor antagonist; OKY-046, a thromboxane A2 synthase inhibitor; nicardipine, a Ca(2+)-channel antagonist and by Ca(2+)-free solution. In addition, spontaneous electrical activity was enhanced by a thromboxane A2 analogue and by prostaglandin H2. Spontaneous electrical activity was unaffected by phentolamine, atropine and tetrodotoxin. 4. Membrane potential in arteries of salt-loaded DS rats was not affected by either indomethacin or ONO-3708. 5. Spontaneous contraction, sensitive to indomethacin, was present, and contractile sensitivity to high potassium solution was enhanced in arteries of salt-loaded DS rats. 6. These findings suggest that eicosanoid action, together with membrane depolarization, may lead to the activation of voltage-dependent Ca(2+)-channels, thereby causing spontaneous electrical activity in mesenteric arteries of salt-loaded DS rats. In addition, tension data suggest that these changes in membrane properties are related to enhanced contractile activities in salt-loaded DS rats. Mechanisms of depolarization remain to be determined.

Animals↗
PubMed1997

Effect of aging on regulation of brain stem circulation during hypotension.

This study was designed to determine age related changes in autoregulatory responses of the brain stem circulation in vivo. In anesthetized adult (4 to 6 months, n = 8) and aged (24 to 26 months, n = 7) Sprague-Dawley rats, local CBF to the brain stem was determined with laser-Doppler flowmetry and diameters of the basilar artery and its branches were measured through an open cranial window during stepwise hemorrhagic hypotension. In aged rats, the lower limit of CBF autoregulation shifted upward to 60 to 75 mm Hg from 30 to 45 mm Hg in adult rats. Dilator responses of the basilar artery (baseline diameter: 254 +/- 15 microns), large branch (109 +/- 23 microns), and small branch (44 +/- 10 microns) to hypotension were much smaller in aged rats than in adult rats. The maximum change in diameter of the basilar artery during profound hypotension was significantly smaller in aged rats (11 +/- 8%) than that in adult ones (23 +/- 12%, P < 0.05); that of the large branch was 12 +/- 8% versus 33 +/- 17% (P < 0.005); and that of the small branch was 17 +/- 7% versus 40 +/- 13% (P < 0.0005), suggesting greater attenuation of the responses in the smaller vessels. Thus, this study provides direct evidence that aging diminishes the compensatory dilatation of brain stem arterioles and arteries during hypotension and modifies the autoregulatory plateau of CBF, which seems to increase the risk of the brain stem ischemia during hypotensive conditions.

Acetylcholine↗
PubMed1997

Age-related changes in response of brain stem vessels to opening of ATP-sensitive potassium channels.

BACKGROUND AND PURPOSE: This study was designed to determine regional differences and age-related changes in the contribution of ATP-sensitive potassium (KATP) channels to vasodilator responses in the brain stem circulation in vivo. METHODS: Changes in diameter of the basilar artery (baseline diameter, 270 +/- 5 microns [mean +/- SEM]), its large branch (112 +/- 5 microns), and its small branch (49 +/- 2 microns) in response to KATP channel openers levcromakalim and Y-26763 were measured through a cranial window in anesthetized adult (4 to 6 months) and aged (24 to 26 months) Sprague-Dawley rats. RESULTS: Topical application of levcromakalim and Y-26763 produced concentration-dependent vasodilation that was similar among the three vessel groups in adult rats. In aged rats, dilator responses of the branches, but not of the basilar artery, to the KATP channel openers were smaller than those in adult rats (P < .05). Glibenclamide, a selective KATP channel blocker, almost abolished this vasodilation in both groups of rats. Vasodilator responses to sodium nitroprusside were preserved in aged rats. CONCLUSIONS: In adult rats, there is no regional heterogeneity in vasodilator response to KATP channel openers in the brain stem circulation in vivo. In aged rats, although KATP channels are also functional in the brain stem circulation, dilator response of the microvessels but not of the large arteries to direct activation of KATP channels is impaired.

Aging↗
PubMed1997

Evidence for functional roles of Crk-II in insulin and epidermal growth factor signaling in Rat-1 fibroblasts overexpressing insulin receptors.

We examined the potential role of Crk-II in insulin and epidermal growth factor (EGF) signaling in Rat-1 fibroblasts overexpressing insulin receptors. Crk is an SH2 and SH3 domain-containing adaptor protein that has been reported to associate with p130cas, paxillin, c-cbl, c-abl, Sos, and C3G in vitro. Insulin- and EGF-induced association of Crk-II with these molecules was assessed by immunoblotting of anti-Crk-II precipitates in Rat-1 fibroblasts overexpressing insulin receptors. Neither insulin nor EGF treatment induced Crk-II association with either Sos or C3G. Basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin or EGF. p130cas and paxillin were heavily tyrosine phosphorylated in the basal state. Both insulin and EGF stimulated their dephosphorylation, followed by p130cas-Crk-II dissociation and paxillin-Crk-II association, although the magnitude of these effects was greater with insulin than with EGF. Interestingly, EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. To investigate the functional roles of Crk-II in mitogenesis and cytoskeletal rearrangement, we performed microinjection analysis. Cellular microinjection of anti-Crk-II antibody inhibited EGF-induced, but not insulin-induced, DNA synthesis. Insulin, but not EGF, stimulated cytoskeletal rearrangement in the cells, and microinjection of anti-Crk-II antibody effectively inhibited insulin-induced membrane ruffling, suggesting that Crk-II is involved in insulin-induced cytoskeletal rearrangement. These results indicate that Crk-II functions as a multifunctional adaptor molecule linking insulin and EGF receptors to their downstream signals. The presence of c-cbl-Crk-II association may partly determine the signal specificities initiated by insulin and EGF.

Animals↗
PubMed1997

[Improvement of longitudinal resolution by deblurring technique in helical CT of the head and neck].

We assessed the improvement of longitudinal resolution in helical CT by deblurring techniques in a phantom and 8 head and neck patients. After helical scanning with 3- or 5-mm-collimation and 3- or 5 mm/sec table feed, overlapped axial images were reconstructed at every 0.3 or 0.5 mm pitch. Deblurring techniques were applied to these data. The slice profile and longitudinal resolution were improved by deblurring. Longitudinal resolution increased with the strength of deblurring. Image sharpness was improved in reconstructed images of MPR, MIP and 3D, and better visualization of the peripheral vessels was obtained in CTAs.

Head and Neck Neoplasms↗
PubMed1997

Chronic administration of aprindine during maintenance hemodialysis.

Aprindine was administered for 12 months to 8 hemodialysis patients suffering from arrhythmias. The serum aprindine concentration ranged from 0.3 to 0.6 microgram/ml, and did not increase with time during the 1-year treatment period. The PQ interval was temporarily prolonged in the first and second months, but the QRS and QT intervals were not changed by chronic aprindine treatment. The changes of the PQ interval in the second month of treatment were directly correlated with the serum aprindine concentration. No alterations of the ECG findings were observed when aprindine was discontinued. The cardiothoracic ratio (chest radiography) and laboratory findings were also not influenced by either aprindine treatment or its withdrawal. In conclusion, aprindine may be safely administered for at least 1 year to arrhythmia patients on maintenance hemodialysis.

Administration, Oral↗
PubMed1996

Crystal structure of glycine N-methyltransferase from rat liver.

Glycine N-methyltransferase (GNMT) from rat liver is a tetrameric enzyme with 292 amino acid residues in each identical subunit and catalyzes the S-adenosylmethionine (AdoMet) dependent methylation of glycine to form sarcosine. The crystal structure of GNMT complexed with AdoMet and acetate, a competitive inhibitor of glycine, has been determined at 2.2 A resolution. The subunit of GNMT forms a spherical shape with an extended N-terminal region which corks the entrance of active site of the adjacent subunit. The active site is located in the near center of the spherical subunit. As a result, the AdoMet and acetate in the active site are completely surrounded by amino acid residues. Careful examination of the structure reveals several characteristics of GNMT. (1) Although the structure of the AdoMet binding domain of the GNMT is very similar to those of other methyltransferases recently determined by X-ray diffraction method, an additional domain found only in GNMT encloses the active site to form a molecular basket, and consequently the structure of GNMT looks quite different from those of other methyltransferases. (2) This unique molecular structure can explain why GNMT can capture folate and polycyclic aromatic hydrocarbons. (3) The unique N-terminal conformation and the subunit structure can explain why GNMT exhibits positive cooperativity in binding AdoMet. From the structural features of GNMT, we propose that the enzyme might be able to capture yet unidentified molecules in the cytosol and thus participates in various biological processes including detoxification of polycyclic aromatic hydrocarbons. In the active site, acetate binds near the S-CH3 moiety of AdoMet. Simple modeling indicates that the amino group of the substrate glycine can be placed close to the methyl group of AdoMet within 3.0 A and form a hydrogen bond with the carboxyl group of Glu15 of the adjacent subunit. On the basis of the ternary complex structure, the mechanism of the methyl transfer in GNMT has been proposed.

Amino Acid Sequence↗
PubMed1996

Functional importance of amino-terminal domain of Shc for interaction with insulin and epidermal growth factor receptors in phosphorylation-independent manner.

Shc has two distinct domains, amino-terminal and SH2 domain, which can interact with activated growth factor receptors. Shc interacts with insulin receptor via Shc-amino-terminal (N) domain, whereas Shc associates with epidermal growth factor (EGF) receptor through both Shc-N and -SH2 domains. In accordance with the different functional roles between insulin and EGF receptors, EGF stimulated tyrosine phosphorylation of Shc faster than insulin. To clarify the functional importance of three distinct Shc domains on insulin and EGF signaling, we microinjected glutathione S-transferase (GST) fusion proteins containing the amino terminus plus collagen homology domain (NCH), collagen homology domain (CH), and Src homology 2 domain (SH2) into Rat1 fibroblasts expressing insulin receptors (HIRc). Bromodeoxyuridine (BrdUrd) incorporation into newly synthesized DNA was subsequently studied to assess the importance of the three distinct domains of Shc. Microinjection of the NCH-GST fusion protein inhibited BrdUrd incorporation induced by both EGF and insulin, whereas microinjection of the SH2-GST fusion protein inhibited EGF, but not insulin stimulation of DNA synthesis. Neither EGF- nor insulin-induced BrdUrd incorporation was inhibited by the CH-GST fusion protein. Following EGF or insulin stimulation, Shc is phosphorylated on single Tyr-317 residue serving as a docking site for Grb2. Microinjection of Shc-N+CH GST fusion protein with Tyr-317 --> Phe replacement (Y317F) also inhibited insulin stimulation of DNA synthesis. Next, we stably overexpressed wild-type Shc or Y317F mutant Shc into HIRc cells. Insulin-induced tyrosine phosphorylation of IRS-1 was compared among the transfected cell lines, since IRS-1 and Shc could competitively interact with insulin receptor. Insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in both WT-Shc and Y317F-Shc cells compared with that in HIRc cells. Furthermore, overexpression of the Shc-SH2 domain or Shc-N+CH domain with Y317F mutation interfered with EGF-stimulated endogenous Shc phosphorylation. These results suggest that the amino terminus domain of Shc is functionally important in insulin- and EGF-induced cell cycle progression and that the phosphorylation of Shc Tyr-317 residue is independent of Shc interaction with these receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Characterization of alpha-1,6-mannosyltransferase responsible for the synthesis of branched side chains in Candida albicans mannan.

A particulate insoluble fraction from Candida albicans NIH B-792 (serotype B) strain cells was obtained as the residue after extracting a 105000 x g pellet of cell homogenate with 1% Triton X-100. Incubation of this fraction with a mannopentaose, Man alpha 1-->3Man alpha 1-->2Man alpha 1-->Man alpha 1-->2Man, in the presence of GDP-mannose and Mn2+ at pH 6.0 gave a branched mannohexaose, [sequence: see text] 6 the structure of which was identified by means of sequential off assignment. However, the enzyme fraction obtained from Candida parapsilosis gave Man alpha 1-->2Man alpha 1-->3Man alpha 1-->2Man alpha 1-->2 Man alpha 1-->2Man under the same conditions. These results demonstrate the finding that the structural difference in the mannans of these two species is due to the presence of alpha-1.6-linked branching mannose units in the C. albicans mannan [Shibata, N., Ikuta, K., Imai, T., Satoh, Y., Satoh, R., Suzuki, A., Kojima, C., Kobayashi, H., Hisamichi, K. & Suzuki, S. (1995) J. Biol. Chem. 270, 1113-1122]. The substrate-specificity study of the enzyme indicated that the structural requirement of the alpha-1,6-mannosyltransferase is Man alpha 1-->3Man alpha 1-->. The alpha-1,6-mannosyltransferase also transferred the alpha-1,6-linked branching mannose unit to the mannan of Saccharomyces cerevisiae. The transformation of the mannan was detected by the appearance of antigenic factor 4 using an enzyme-linked immunosorbent assay and two-dimensional homonuclear Hartmann-Hahn spectroscopy.

Candida↗